1. Field of the Invention
This invention resides in the field of biological binding assays, and relates in particular to the transfer of binding members or other reagents to spots, bands, or other spatial arrays on a membrane, gel, or other planar support for purposes of detection, identification, and quantification.
2. Description of the Prior Art
Proteins, nucleic acids, or other biological species that have been electrophoretically separated in a slab gel are often transferred to a membrane of nitrocellulose, nylon, polyvinyl difluoride, or similar materials for identification and quantification since these steps are more easily performed on the membrane than in the gel. A common transfer technique is electroblotting, in which the flat surfaces of the gel and membrane are placed in direct contact and an electric current is passed through both the gel and the membrane in a transverse direction, thereby transferring the species in a manner similar to that by which the species were mobilized within the gel. When the species are DNA fragments, the transfer is termed a Southern blot after its originator, the British biologist Edwin M. Southern. By analogy, the transfer of RNA fragments is termed Northern blotting, and the transfer of proteins or polypeptides is termed Western blotting. Still further examples are “Eastern” blots for post-translational modifications, and “Far Western” blots for protein interactions.
Electroblotting of all of these types can be performed in either a wet, dry, or semi-dry format. In wet blotting, the gel and membrane are layered over each other in a stack which is immersed in a transfer buffer solution in a tank on whose walls are mounted wire or plate electrodes. The electrodes are then energized to cause the solutes to migrate from the gel to the membrane. In semi-dry blotting, filter papers wetted with the transfer buffer solution are used, and the stack contains the filter papers on the top and bottom with the gel and the membrane between the filter papers to form a “blotting sandwich.” The electrodes are then placed in direct contact with the exposed surfaces of the wetted filter papers. Dry electroblotting uses no liquid buffers other than those residing in the gels. Descriptions of wet, dry, and semi-dry electroblotting and the associated materials and equipment are found in Margalit et al. (Invitrogen) United States Patent Application Publication Nos. US 2006/0272946 A1, published Dec. 7, 2006, US 2006/0278531 A1, published Dec. 14, 2006, and US 2009/0026079 A1, published Jan. 29, 2009; Littlehales (American Bionetics) U.S. Pat. No. 4,840,714, issued Jun. 20, 1989; Dyson et al. (Amersham International) U.S. Pat. No. 4,889,606, issued Dec. 26, 1989; Schuette (Life Technologies, Inc.), U.S. Pat. No. 5,013,420, issued May 7, 1991; Chan et al. (Abbott Laboratories), U.S. Pat. No. 5,356,772, issued Oct. 18, 1994; Camacho (Hoefer Scientific Instruments), U.S. Pat. No. 5,445,723, issued Aug. 29, 2005; Boquet (Bertin & Cie), U.S. Pat. No. 5,482,613, issued Jan. 9, 1996; and Chen (Wealtec Enterprise Co., Ltd.) U.S. Pat. No. 6,592,734, issued Jul. 15, 2003.
Regardless of whether electroblotting is performed in a wet, dry, or semi-dry format, the resulting electroblot is further treated with detection reagents to render the biological species in the blot detectable by methods appropriate to the species themselves. In Southern and Northern blots, for example, the detection reagents are hybridization probes monitored by detection of a fluorescent or chromogenic dye. In Western blots, the detection reagents can include primary antibodies followed by the use of a secondary antibody labeled with a fluorophore, a chromophore, or an enzyme to detect the antibodies. Alternatively, the secondary antibody can be labeled with a detection agent such as biotin or avidin/streptavidin, and the presence or absence of the detection agent can be detected. Similar or analogous procedures, known among skilled biochemists, are performed with Far Western blots and Eastern blots.
Spatial arrays other than electroblots are also used in biological assays. Binding assays can be performed directly on slab gels, for example, as well as mass spectroscopy targets, ELISA plates, and on proteins, nucleic acids, or other biological species that have been deposited on a membrane or other support surface in regularly spaced or irregularly spaced two-dimensional arrays by electrospraying, vacuum deposition, pin spotting, and other methods.
In all of these spatial arrays, the binding reagents that are applied to the spots, including detection reagents, hybridization probes, antibodies, or other species, are a significant component of the cost of these procedures, and much of the volume of any given reagent is wasted since the bands or spots where binding is to occur occupy but a small fraction of the surface area of the support. One way to limit the cost is to use a highly diluted form of the binding reagent, but this requires a prolonged contact time which places a limit on the number of analyses that can be performed in a given period.